The goal of this research is to develop novel, more effective therapies for the treatment of tuberculosis, particularly directed towards the development of new therapies for treating latently-infected patients. To accomplish this goal we must first understand the metabolic processes which are associated with latency, particularly with respect to identifying key physiological processes which facilitate long-term survival of tubercle bacilli in the absence of detectable growth. Initially we sought to characterize the metabolic state of the organism when grown in vitro for extended periods of time. We used two-dimensional gel electrophoresis to examine both the steady-state protein composition and time-dependent protein synthetic profiles in aged cultures of virulent M. tuberculosis (MTB). At least seven proteins were identified which were differentially expressed as cultures went from log phase into stationary phase. One of these proteins, which was not detectable in log phase protein profiles, accumulated to become the single most abundant protein in stationary phase organisms. N-terminal sequence analysis and subsequent immunoreactivity were used to identify this protein as the 16kDa alpha-crystallin antigen which had been previously identified as a major immunogenic component associated with the cell wall. Cloning the gene for this protein allowed us to assertain that transformed MTB, overexpressing this protein, showed a slower loss of viability following saturation of long-term cultures in vitro. Overexpression of this protein and subsequent purification allowed functional assays based upon the suppression of thermal denaturation of a variety of unrelated proteins. The gene for this protein was limited to pathogenic mycobacteria and appears to be regulated by oxygen concentration in the media but not by a wide variety of other environmental factors. Gene fusions to a luciferase reporter allowed us to demonstrate that the alpha crystallin promoter is induced 1000-fold upon shifting the oxygen concentration from 5% to 1.3% in a matter of hours. The gene for this protein was used to construct a genetic knockout in virulent MTB and this strain was used to assess growth rates in vitro and in vivo. In vitro growth was normal under high oxygen conditions and somewhat impaired upon oxygen restriction. Preliminary experiments show the knockout is significantly less virulent in infections of macrophage cell lines and primary mouse bone marrow-derived macrophages. Preliminary mouse challenge studies demonstrate differences in infectivity with the knockout being impaired for growth in mice.
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